Trypanosoma cruzi, (T. cruzi) a hemoflagellate, is the causative agent of Chagas' disease. Chagas' disease is a major health problem in Central and South America, especially Mexico, Brazil, Chile and Argentina. It is currently estimated that at least 12 million individuals are infected with the protozoan parasite (Hudson, 1981). In addition, there have been occassional reports of occurrence of Chagas' disease in California and Texas.
Chagas' disease principally occurs in rural areas and correlates with poor housing and sanitary conditions. Houses are excellent breeding places for the insect vector, a common blood sucking parasite (reduviid sps. Triatoma, Panstrongylus and/or Rhodnius), which transmits the disease between vertebrate hosts, such as man, domestic pets including cats and dogs, and wild mammals.
When an insect host, containing infectious metacyclic trypomastigotes in its hindgut, defecates onto the skin of its victim while feeding and engorging on the victim's blood, the T. cruzi trypomastigotes are deposited on the victim. These trypomastigotes can then easily penetrate the mucosae once the skin has been broken by wound, abrasion, etc. The simple act of scratching the bite area facilities entry of the parasite into the victim.
T. cruzi exists in a number of different morphological forms. Epimastigotes and metacyclic trypomastigotes are normally found in the insect midgut and hindgut, respectively. Blood form trypomastigotes are found in the circulation of infected vertebrates. The amastigote lives and reproduces inside vertebrate host cells. Epimastgotes have been the normal source of diagnostic antigens since they can be grown cheaply in axenic culture. Although it is believed that the trypomastigotes would be a superior source of antigens, their high cost of production and the higher risk of infection has generally precluded their use.
The progression of Chagas' disease is divided into two phases. In the initial acute phase there is a parasitemia present accompanied by mild flu-like symptoms or no apparent symptoms at all. After approximately two weeks the host's immune system clears the circulating parasites and the chronic phase is established. During the chronic phase the parasites exist intracellularly thereby escaping immune surveillance. After several years the symptoms of the chronic phase of Chagas' disease may appear, including myocardia enlargement, hardening of the esophagus and/or lower intestine, and neuromotor problems. Autoantibodies to neural and muscular antigens are usually associated with the chronic form of Chagas' disease. Chagas' disease is especially insidious because there are no satisfactory prophylactic or curative agents available and because an individual, once he has contracted the disease, remains infected for life.
The only useful diagnostic parameters during the final stage of the disease are either parasitological or immunological testing, due to the variability or possible lack of specific clinical symptoms. Parasitological diagnosis during the chronic phase is expensive, time-consuming and often extremely distasteful to the patient. Because circulating parasites cannot be detected by direct examination of blood smears, parasitological testing (xenodiagnosis) must be employed. In such tests, reduviid bugs are permitted to feed on the patient, and the hindguts of the reduviid bugs are examined under microscope 30 days after such feeding to determine if T. cruzi is present.
The preferred and more widely used tests for Chagas' disease detection are immunological (serological) analysis, including immunofluoresence (IF); complement fixation (CF); either direct (DA) or indirect hemagglutination (IHA); latex agglutination (LA) and enzyme-linked immunoassay (ELISA).
However, the current serological tests are unsatisfactory for a number of reasons. First, a large variation exists between these tests; for example, a serum giving a high titer from one test frequently gives a low or moderate titer in another test. Due to this variation, three different serological tests must generally be performed in parallel and at least two of them must give high titers for positive diagnosis. Second, production of the diagnostic reagents is hazardous because the parasites must be grown and handled to produce the antigen extracts. Third, use of crude mixed antigen preparations (basically fixed epimastigotes) causes cross-reactivity with patients who have Leishmania (another endemic parasitic protozoan), so that many false positives are indicated. Fourth, at least some of the other test methods require the use of sophisticated laboratory equipment not generally available or practical in the geographic areas with a high incidence of Chagas' disease.
The development of a sensitive and accurate diagnostic test for Chagas' disease has a high priority, particularly in Central and South America, where over 200 million individuals are at risk of infection. To facilitate this goal it is necessary to identify and isolate unique T. cruzi proteins which are specifically recognized by the serum from chagasic patients.
The literature on T. cruzi contains conflicting data concerning immunoreactive surface proteins and glycoproteins. Many researchers have described glycoproteins in the 70 kd 95 kd region, yet it is not clear which specific proteins the different reports are describing. The cell surface components of several morphological forms of T. cruzi have been radiolabeled and analyzed by one and/or two-dimensional polyacrylamide gel electrophoresis (1D- or 2D-PAGE). A 72 kd surface protein has been identified on epimastigotes and metacyclic trypomastigotes [Snary and Hudson, (1979); Nogueira et, al, (1981); Snary et al, (1981); Zingales et al, (1982); and Lanar and Manning, (1984)). A 90 kd surface protein has been identified in bloodstream and tissue culture-derived trypomastigotes Snary and Hudson, (1979); Araujo and Remington, (1981); Nogueira et al, (1981); Snary et al, (1981); Zingales et al, (1982); Nogueira et al, (1982); and Lanar and Manning, (1984)]. In addition, Snary and Hudson, (1979) and Zingales et al, (1982) have reported the 90 kd protein on epimastigotes, although Nogueira et al, (1982); Manning (1984) and Dragon et al, (1985) have not seen this. Katzin and Colli (1983) have identified an 85 kd trypomastigote specific protein. Lanar and Manning (1984) have determined with radioimunoassays and western blot analysis that the 90 kd protein is one of the major antigenic components recognized by sera from mice chronically infected with T. cruzi. Nogueira et al have shown that IgG isolated from the blood of 5 patients hospitalized in Brazil with chronic Chagas' cardiopathy primarily reacts with proteins in the 90 kd region. Further, a 25 kd protein has also been reported as being cross-reactive with chagasic sera [Scharfstein et al, (1983)].
Thus, there is a need for a detection system for Chagas' disease which provides reproduceable results, and which is relatively easy, safe and inexpensive to manufacture and perform. Recombinant DNA technology is available to accomplish the foregoing objectives.